Antibodies are an important class of pharmaceutical products that have been successfully used in the treatment of various human diseases and conditions, including infectious diseases, cancer, allergic diseases, and graft-versus-host disease, as well as in the prevention of transplant rejection.
One problem associated with the therapeutic application of non-human immunoglobulins is the potential immunogenicity of the same in human patients. In order to reduce the immunogenicity of such preparations, various strategies for the production of partially human (humanized) and fully human antibodies have been developed. The ability to produce transgenic antibodies having a human idiotype in non-human animals is particularly desirable as antigen binding determinants lie within the idiotype region, and non-human idiotypes are thought to contribute to the immunogenicity of current antibody therapeutics. Human idiotype is an especially important consideration in respect of monoclonal antibody therapeutics, which consist of a single idiotype delivered at relatively high concentration as opposed to the variety of idiotypes delivered at lower concentrations by a polyclonal antibody mixture.
While a number of approaches to producing humanized transgenic antibodies in non-human animals have been described, one major problem encountered in many such approaches is the production of endogenous antibody, either preferentially or in combination with transgenic antibodies in the host animal. Various recombinant cloning schemes have been used in attempts to disrupt endogenous immunoglobulin production in host animals to address this problem. However, the functional inactivation of immunoglobulin genes presents many obstacles in many vertebrate species.
For example, while homozygous mutant mice with deleted JH-loci have been successfully produced using homologous recombination, ES or other sustainable pluripotent cells in which homologous recombination can be done to inactivate endogenous loci are not readily available from most vertebrate species.
Further, mutations that interfere with cell surface expression but not with productive rearrangement of immunoglobulin VDJ or VJ gene-segments are insufficient to inactivate endogenous Ig expression completely. This is exemplified by the fact that homozygous mutant mice with a disrupted membrane exon of the μ heavy chain (so called μMT mice) cannot produce IgM or IgG, but still produce significant quantities of IgA (Macpehrson et al. Nature Immunol 2(7):625-631 (2001). In addition, the serum of heterozygous mutant mice contains IgM and IgG encoded by both alleles, the wild-type allele and the mutated μMT allele (Kitamura and Rajewky, Nature 356:154-156 (1992). This is due to the fact that the first rearrangement in the course of B-cell development is the joining of DH- and JH-gene segments on both homologous chromosomes, generating a pro-B cell. If, in the μMT/+ mice, a pro-B cell undergoes subsequent VH-DHJH joining in the mutated IgH locus first and the joining is in frame (“productive”), the resulting pre-B cell can express a μ chain of the secreted form, but cannot express membrane-bound μ Since membrane-bound μ expression is required for allelic exclusion, such a cell is still able to undergo VH-DHJH joining in the wild-type IgH locus; and if this second rearrangement is also productive, the cell expresses two different μ chains, one of which is membrane-bound. Serum of such mice contains IgM derived from both alleles. In addition, IgG derived from both alleles can be found in the serum of such mice because switching is often concomitantly induced on both IgH loci of a B cell.
Incomplete allelic exclusion is also observed in animals with functional transgenic immunoglobulin loci and mutated endogenous immunoglobulin loci that can still rearrange VDJ or VJ gene segments productively. A B-cell rearranging VH-DHJH in one or both mutated endogenous loci may still rearrange transgenic immunoglobulin loci productively. Such a B-cell expresses membrane-bound transgenic immunoglobulin and develops into a mature B-cell. During B-cell development isotype switching in the mutated endogenous locus may result in a B-cell expressing endogenous immunoglobulin. Accordingly, such mutations are insufficient for the complete inactivation of endogenous immunoglobulin expression in animals with transgenic immunoglobulin loci.